Murine leukaemia virus--derived retroviral vectors are being used in clinical gene therapy trials for ex vivo transduction of T cells, hepatocytes, haemopoietic stern cells, synoviocytes, fibroblasts and a variety of neoplastic cells (Human gene marking/therapy clinical protocols, 1994 Hum. Gene Ther. S p417-426: Clinical protocols. 1994 Cancer Gene Ther. I p73-78). However, low frequency of target cell transduction is proving to be a serious limiting factor in these early trials (Rosenberg et al. 1990 N. Engl. J. Med. 323 p570-578; Brenner et al, 1993 Lancet 341 p85-88; Grossman et al, 1994 Nat. Genet. 6 p335-341; Rill et al, 1994 Blood 84 p380-383).
The efficiency of retroviral gene transfer is influenced by a number of factors including the concentration and physical integrity of the virus particles and the concentration, proliferative activity and inherent susceptibility of the target cells (Vile & Russell, 1995 Brit. Med. Bulletin 51 p12-30). Local conditions affecting the kinetics of virus adsorption to target cell membranes also have a major influence on retroviral titres. Polybrene is used routinely to enhance the adsorption of retroviral vectors and usually increases the titre several-fold. However, during 2-hour incubations of MLV-derived retroviral vectors with target cells in standard conditions (i.e. with added polybrene) only a small percentage (ea. 5%) of the infectious various were depleted from the medium indicating that virus adsorption is highly inefficient (i.e. slow rate of adsorption) even in the presence of polybrene (Wang et al, 1991 J. Virol, 65 p6468-6477). The kinetics of virus adsorption are likely to be equally slow for other viruses in similar ex vivo tissue culture systems. Low speed centrifugation of unprecipitated retroviral vectors onto their target cells has been shown to enhance transduction efficiencies, presumably by speeding the rate of specific virus adsorption (Kotani et al, 1994 Hum. Gene Ther. S p19-28).
Chromatography on calcium phosphate (hydroxyapatite) columns has previously been used to purify and concentrate proteins (Tiselius et al, 1956 Arch. Biochem. Biophys. 65 p132-155), DNA (Main & Cole, 1957 Arch. Biochem. Biophys, 68 p186), RNA (Semenza, 1957 Biochim. Biophys. Acta 24 p401) and viruses (Taverne et al, 1958 I. Gen. Microbiol. 19 p451-461).
Influenza virus purification has also been achieved by adsorption on a precipitate of calcium phosphate formed by adding calcium chloride to a phosphate-containing solution (Salk. 1941 Proc. Soc. Exp. Biol. Med. 46 p709-712; Stanley, 1945 Science 101 p332-335). To purify the virus, the washed precipitate was redissolved in citric or hydrochloric acid and dialysed against distilled water. There was little, if any, loss in virus infectivity during the process of washing, elution and dialysis (Salk, 1941 Proc. Soc. Exp. Biol. Med. 46p709-712). To secure the most effective precipitation of virus, it was necessary to carry out the formation of the calcium phosphate precipitate in the presence of the virus (Stanley, 1945 Science 101 p332-335). The infectivity of viruses still bound to calcium phosphate was not tested and there are no reports of attempts to purify retroviruses in this way. Also, there are no reports of attempts to enhance viral gene transfer by presenting viruses to cells as a complex with precipitated calcium phosphate.
DNA adsorbs to calcium phosphate formed when calcium chloride is added to a phosphate-containing solution and this provides the basis of a widely used technique for introducing foreign DNA into mammalian cells (Graham & van der Eb, 1973 Virology 52p456-467). The mechanism of DNA uptake by this method has been studied in detail using fluorescent dyes to independently follow the fates of the DNA and of the calcium phosphate (Loyter et al, 1982 Proc. Natl. Acad, Sci, USA 79 p422-426). The DNA forms a tight complex with the calcium phosphate and the DNA in the complex is resistant to nucleases. The calcium phosphate increases the concentration of DNA on the cell surface by precipitation, induces endocytosis, facilitating entry of DNA into cells, and protects the DNA from degradation by intracellular nucleases. Cellular uptake of the calcium phosphate--DNA complexes is highly dependent upon the pH at which they are formed and upon the concentration of DNA in the complex. However, only a small proporation of the DNA that has been taken up by the cell moves from the cytoplasm to the nucleus. Hence the efficiency of gene expression after DNA transfection remains very low.
Molecular conjugate vectors were developed to overcome some of the limitations of previous nonviral gene delivery systems. The major limitation with calcium phosphate transfection was the inefficiency with which DNA delivered as a calcium phosphate co-precipitate could escape from endosomal vesicles into the cytosol. In molecular conjugate vectors, receptor-mediated endocytosis of the DNA is achieved by complexing it to a macromolecular ligand and escape from the endosome is achieved by adding an endosomolytic agent to the complex, such as an adenovirus particle (Michael & Curiel, 1994 Gene Therapy I p223-232).
The endosomolytic properties of adenovirus particles have also been harnessed to facilitate gene transfer by retroviral vectors to cells outside of their normal host range (Adams et al 1995 J. Virol, 69 p1887-1894). In the presence of adenovirus, several different ecotropic retroviral vectors were shown to infect human cell lines and a xenotropic vector was shown to infect marine cells. However, in these experiments there was no attempt to achieve physical linkage between the adenovirus and retrovirus particles before they were placed in contact with the target cells.